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1961 (E)

[00:00:00] Here we are with Book E, starting in early 1961. The initial experiments have a clear motivation. The aim was to reduce and prevent reformation of disulfide bonds, to enable conditions to be established for 1‑1 and 2‑2 maximum SH. By that I mean depolymerization without complete wreckage. And being careful to avoid reoxidation, by using Versene. That was the trade name for EDTA. (laughs) The comment [00:01:00] the result of trying that type of experiment was disappointing result, as judged by the urea, formate gel. “The 1‑1 is very resistant to mercaptoethanol.” And the 2‑2… Let’s see what it says about the 2‑2. “A possible ‘monomer,’” in quotes, “of 2‑2 at 0.05-molar.” But I wasn’t very sure about this, to try a direct procedure, once again, with haptoglobin 1‑1 and 2‑2. In other words, I wasn’t getting any change that was obvious, with haptoglobin 1‑1, but I could get changes with the haptoglobin 2‑2. [00:02:00] Pursuing this idea. Synthesis of a cross-linking agent, iodoacetamide combined with [CH2=CH2­?]. I don’t know why I was wanting to make this one. Or for hemoglobin dimerization or polymerization. “Look up [bifunctional?] mercury compounds,” etc. So I was trying some organic chemistry.

Page 7, Saturday, February 4, different concentrations of mercaptoethanol, and [00:03:00] adding iodoacetamide, to try to capture a depolymerized product. Gels not very convincing. The aim was to establish an equilibrium, with enough mercaptoethanol to open up [the?] 2‑2 for reformation of 2‑1. Again, a mixture of 2‑1 and 2‑2 — or 1‑1 and 2‑2 — mixtures, trying to make the heterozygote from the two homozygote. Page 9, the result’s not very good. Very slight possibility that some synthesi‑‑ is taking place, with mercaptoethanol but not with mercaptoethanol plus iodoacetamide. [00:04:00] Pursuing this idea. Splitting in the presence of hemoglobin tried, on February 14th. And rough tests of precipitated material. A comment that “The hemoglobin-haptoglobin appears to be OK. It’s the hemoglobin which is wrecked.” “Very marked effect of mercaptoethanol on 2‑2 hemoglobin,” on Wednesday, February 15th — 13th — oh, 15th, page 13.

[00:05:00] (long pause) February the 16th, Thursday, where material that had been treated — 1 milligram of 1‑1, 2‑1, and 2‑2, with deionized hemoglobin, and then EDTA. Mercaptoethanol added. And then run on a borate [00:06:00] gel, containing the sodium sulfur — Versene. “The results are confusing,” is the comment. Clearly, the 2‑1 has dissociated and so has the 2‑2. And something is running… And — yeah. Do that again. The comment on page — 1/16th is, “The results are confusing. Cannot recognize hemoglobin-haptoglobin. And yet the polymers are abolished.” So something is working, in terms of getting rid of the polymers, in the presence of [00:07:00] EDTA. And so on.

Testing on other gels, February 17th, in the presence of EDTA, mercaptoethanol gels containing different amounts of mercaptoethanol. New 2‑2 is split OK. The faster components are haptoglobin products. And it looks clean. So I was somewhat encouraged. Although on page 21 I say, “These results are surprising. [00:08:00] Even at 0.01-molar mercaptoethanol, multiples are visible.”

So I can try for resynthesis — which I attempted on Saturday, February 18th, resynthesis tests in the presence of Versene — with a co‑‑ This is haptoglobin 1‑1, 2‑1, and a mixture again, and 2‑2, and treated in the presence of hemoglobin, and run in a — in a regular borate gel, containing [00:09:00] Versene, and one also containing mercaptoethanol. With a comment that, “If unsuccessful but promising, repeat with hemoglobin.” Looking at the gels, it’s not clear exactly what’s happening. Does appear to be some destruction — or some depolymerization going on. And, in fact, it says on page 25, following from page 23 “– repeat with hemoglobin” — over the page, then, “Indication that the reaction is incomplete in [00:10:00] 60 minutes. Therefore made the second gel, without mercaptoethanol, for a repeat test.” And again, the comment here on something previously said, “Looking at the [E‑23?] gel suggests that 1‑1 is much tougher to open up than 2‑2. May have to use — have to use different conditions for 1‑1.” (long pause)

Page 27, [00:11:00] interpretation of the results are that “2‑1 and 2‑2 are completely depolymerized at 0.01-molar mercaptoethanol.” But when run in the absence of mercaptoethanol, get the normal patterns. Still finding 1‑1 was behaving differently. This eventually, I know, led to a paper on the depolymerization and repolymerization, when I really understood the reaction. But at this state, I haven’t yet understood the reaction well enough to make is useful.

(laughs) Oh, the comment on page 29, [00:12:00] an attempt at resynthesis, “Results show 1‑1 resynthesis is virtually complete but 2‑1 less and 2‑2 only slightly.” So different temperatures, attempting to get this reversibility. Temperature and pH test on page 31 — which are commented on page 33 by a big oval, saying, “See page 37.” So we’ll look forward to page 37. “Sulfur reaction was reconsidered,” on Monday, February 20th, page 35, with a comment, “No change.” [00:13:00] And now we’ve reached page 37. Let’s see what it says. “Review of the [E-prime-33?] experiment suggests that at pH 8.6, at 0.05-molar mercaptoethanol, there could be a trace of 2‑1 formed, at 37°.” Trying to think of repolymerizing 2‑1. “Consider using hydroxylamine,” to get the SCO bonds, with a comment on the reaction that might be used.

[00:14:00] Thiol-ester bonds, February 22nd, Wednesday. “Beginning to think that 1‑1-to-2‑2 bond differs from the intra-2‑2 and is not a [S‑S?] bond.” That is an unfortunate digression in thought. But the tests with hydroxylamine were quite clear, that — on page 41 — with or without hydroxylamine, in the presence of [V40?] Versene gel, 0.01-molar hydroxylamine, [00:15:00] with or without mercaptoethanol, that the result is that the — that the hydroxylamine’s apparently without effect. “But do one experiment with hydroxylamine and mercaptoethanol, to see.” Thursday, the 23rd, doing that type of experiment.

And a whole bunch of different things were being tested, pH 6.2 to pH 9.8, 0.01-molar mercaptoethanol, etc., urea, mercaptoethanol, f‑‑ sodium sulfide [00:16:00] and mercaptoethylamine and cyanide and hydroxylamine and mercaptoethanol, etc. And, “These samples are also completely alike. Better check splitting with a dilute Versene gel,” etc., etc. So everything looked alike. Not very much progress. More urea and cyanide, on the 24th. Various repeats, on the 29th. Beginning to think I was using up my material purified. Because on page 49 I see, “Check the possibility of doing these experiments with crude haptoglobin, [00:17:00] from DEA elution, with ammonium acetate. Also try 8‑molar urea, w‑‑ mercaptoethanol, followed by Sephadex,” etc. High-pH tests, on February 26.

Big discussion of things, on Sunday, February 27. “The negative 2‑1 synthesis under all these conditions, the ready 2‑2 resynthesis, and the absence of any 2‑2 in 2‑1 all suggest that the Hp 1 and Hp 2 bond in 2‑1 is quite different from that in 2‑2.” Etc., etc. “If this hypothesis is correct,” so-and-so should happen.

Full-scale tests [00:18:00] with urea, on February 28. (pause) Page 59, “The results show a trace, about 5% yield, of 2‑1, no 2‑2, but plenty of 1‑1. The synthesis is possible.” Trying different pHs. Trying acetone. Urea test at a higher pH, pH 7. Repeat of acetone, on February [00:19:00] 28. Check of mercaptoethanol in the gels. (pause) So going back and looking at them, on page 75, different attempts at resynthesis.

Then, on March 2nd, I made hydroxyethyl disulfide. And I’m beginning to get towards what worked. So [00:20:00] this is HOCH2CH­2­SSCH2CH2OH which I wanted to make. Later on, I think I found I could buy it. The disulfide. But this eventually was the secret, using an excess of the disulfide, in the presence of a trace of the SH compound, and making mixed disulfides. But I haven’t reached that point, in this, yet, in these experiments. “Urea alone. Test a new disulfide. Synthesize,” March 2nd. I was thinking that, by using the disulfide, I could reverse the reaction. In fact, it turned out that it would make the reaction go [00:21:00] further [to completion?].

Complex gel, on page 81. (pause) Beginning clue to what’s happening. “The pH 6 test –” this is page 81 — “the pH 6 test is rather odd. The first 2‑1 band is very clear and 1‑1 is much reduced. But a heavy band has formed at a position a little faster than 2‑2/first but different in diffusion from the usual 2‑1 band in [00:22:00] that position.” Beginning to see some different end product. Reruns of this, on page 83. (pause) More SS tests, on Friday, March the 3rd. Test of the concentration of the mercaptoethanol disulfide, [00:23:00] on Saturday, March 4. (pause) It’s getting close but not there yet. Sunday, March 5th, “Consideration of secondary 2‑2 reaction.” A [synification?] test — abandoned. (pause) [00:24:00] Retest of acetone, at 25%, Monday, March 6. And these, all the polymers have gone, with these tests with acetone. All with hemoglobin added. Indication that hemoglobin might be binding to the haptoglobin 2‑2 that has been depolymerized. But I doubt it. [00:25:00] (pause)

Now the better terminology for the dithiodiglycol — easier compound to understand. “Aldrich product gives a cloudy aqueous solution. Made up a 1‑molar solution and deionized it. I could buy the compound from Aldrich. Which is the beginning of getting towards the end of understanding.

Friday, March 11th, still trying 25% acetone. [00:26:00] “The results show that 1‑1 acetone has no visible 2‑1 at this stage,” etc. Comments on what’s happening. Cold acetone, on Sunday. I still haven’t given up on acetone. March 13th, page 105. Usual sort of comment, on 107, “The results of [B‑105?] are odd. The 2‑2 without acetone shows scarcely any polymers. Must be poor stuff,” and, “With cold acetone, is no different. Thus the evidence suggests that cold acetone is [safe?] but this 2‑2 is no good. Test with good 2‑2.” [00:27:00] But not necessary, because this has been done.

So a full-scale cold test with 25% acetone, on Monday, March 13th. (pause) With not very much indication of anything much happening, from looking at the gels here. But no comment made, at this point. Except on page 111 it says, “The results show that 1‑1 and 2‑2 is not going to 2‑1 in cold acetone.” So you can’t get 1‑1 and 2‑2 to give rise to heterozygous pattern, with this procedure. [00:28:00] On page 111 again, “Possible trace of 2‑1 in the 1‑1 plus 2‑2 but mainly just the mixture of 1‑1 plus 2‑2.” Repeated on the following day — with Versene. And also looking at the effect on transferrin and ceruloplasmin, with a comment that “Ceruloplasmin is unaffected by the Versene, except that the migration is improved.” With transferrin solutions — from… The transferrin was from [Schultz?], via Dave Poulik, MDP, and the ceruloplasmin was [00:29:00] from MDP, Dave Poulik. So these were purified materials tested to see if thousandth-molar myrcene would have any effect on… (pause)

Some tests of Dowex. Not very satisfactory. “No sign of haptoglobin or [new albumin?], except in fraction 63.” Continuing the same sort of vein, page 125. “Still no [00:30:00] appreciable haptoglobin.” These are Dowex experiments. (pause) In the column fractions, on 129, it says, “Haptoglobin in the bulk” sample — “bulk elution but not in the column.” But no images. So next, [that’s?] with Dowex. “Tests of increased plasma-to-Dowex ratio. Results show that haptoglobin is well recovered at [00:31:00] [5R:1P?] ratio. But clear that higher pH introduces some trouble. Repeat with pH adjustment, with acetic acid.” So the Dowex chloride with acetic acid, tested on the next day. And various comments.

Testing in the presence of sodium chloride, on April 19th. “Increasing sodium chloride decreases haptoglobin, at the same rate as albumin. So repeat sodium chloride gradient,” etc. Done again on page 159, “Repeat of the sodium chloride gradient, and Ecteola and DEAE vs. Dowex.” [00:32:00] And with a comment at the bottom that, the yield test, “The yields are all –” the bottom, “The results suggest that the Ecteola [remove?] is approximately equivalent to DEAE. But the purity is almost equal to the Dowex. All haptoglobin in 1 and 2 of each.” So repeated on pools for amounts, with a comment on page 148 that “These are essentially equal with Dowex, only a trace up, if at all.” And that’s the end of this book. [00:32:46]